Adjustments in physiological and morphological traits suggest drought‐induced competitive release of some California plants

Abstract Drought and competition affect how morphological and physiological traits are expressed in plants. California plants were previously found to respond less negatively to resource limitation compared to invasive counterparts. In a glasshouse in Santa Cruz, CA, USA, we exposed five native California C3 grassland species to episodic drought and competition (via five locally invasive species). We hypothesized that leaf morphology would be more affected by competition, and leaf photosynthetic gas exchange more so by drought, consistent with optimal partitioning and environmental filter theories. We expected that traits would exhibit trade‐offs along a spectrum for resource conservatism versus acquisition. Bromus carinatus had greater photosynthetic recovery, while Diplacus aurantiacus had lower percent loss of net assimilation (PLA) and intrinsic water‐use efficiency (iWUE) during drought and competition simultaneously compared to just drought. Stipa pulchra and Sidalcea malviflora gas exchange was unaffected by drought, and leaf morphology exhibited drought‐related adjustments. Lupinus nanus exhibited trait adjustments for competition but not drought. Functional traits sorted onto two principal components related to trade‐offs for resource conservatism versus acquisition, and for above‐ versus belowground allocation. In summary, morphological traits were affected by competition and drought, whereas physiological traits, like leaf gas exchange, were primarily affected by drought. The grassland plants we studied showed diverse responses to drought and competition with trait trade‐offs related to resource conservatism versus acquisition, and for above‐ versus belowground allocation consistent with optimal partitioning and environmental filter theories. Diplacus aurantiacus experienced competitive release based on greater iWUE and lower PLA when facing drought and competition.


| INTRODUC TI ON
Optimal partitioning theory suggests that plants increase biomass allocation to structures that acquire the most limiting resource (Bloom et al., 1985). Stressors can differently affect physiological and morphological traits. Physiological traits are those related to molecular-level interactions of compounds within a plant, whereas morphological traits determine plant shape or structure (Lambers et al., 2008). Water-limited plants have been shown to partition growth more so to root than shoot structures (Liu & Stützel, 2004).
Biotic stressors such as competition can have more varied impacts because it unevenly interacts with abiotic resources, which is further complicated by species-specific responses (Rehling et al., 2021).
Invasive competition could lead to increased allocation to shoots or leaves to increase access to space and light (Pérez-Harguindeguy et al., 2016;Westoby, 1998), or increased allocation to roots to access limiting belowground resources, especially in abiotically harsh systems (Liu & Stützel, 2004;Poorter et al., 2012).
Droughts can lead to shifts in the root-to-shoot ratio (root:shoot) or adjustments in leaf traits related to resource conservative plant strategies (Heckathorn & Delucia, 1996). Plants that are more resource conservative typically grow slower, use less resources, and are more drought resistant, while resource acquisitive species may be more resilient in their recovery from drought or grow fast during wet periods to escape drought (Funk et al., 2008;Kooyers, 2015). Different mixes of acquisitive and conservative traits allow some species to recover from drought (Nicotra et al., 2010), while others may experience unrecoverable physiological stress (Zhong et al., 2019). Photosynthetic rates and biomass allocation are often reduced by drought, and although some species may recover photosynthetic rates fully upon rewetting, others may not (Poorter et al., 2012;Zhong et al., 2019). Certain plants have higher water-use efficiency (WUE) after drought (Lajtha & Marshall, 1994), whereas others have decreased WUE and lower photosynthetic recovery (Zhong et al., 2019) leading to feedbacks that can result in mortality.
Environmental filter theory (Funk et al., 2008) predicts that individuals have to pass through abiotic and biotic filters to establish or sustain co-existing populations at a particular site (Adler et al., 2013).
Abiotic filters like drought often result in different species having similar conservative traits to survive the same harsh micrometeorological conditions. On the other hand, biotic filters facilitate species trait divergence, partitioning of resources, and allowing for species coexistence (Poorter et al., 2012). Passing through abiotic and biotic filters at a particular site may require contrasting values of the same traits (Funk et al., 2008;Pierce et al., 2017). Harsh abiotic conditions and limited resource availability select for resource conservative traits like low specific leaf area (SLA), stomatal conductance (g s ), and growth rates, whereas strong biotic filters associated with competition select for high net CO 2 assimilation (A net ), SLA, and high growth rates (Drenovsky et al., 2012;Pérez-Harguindeguy et al., 2016). Leaf lobedness and vein length can promote trait conservatism by reducing leaf water loss (Cadotte et al., 2015;Sack & Scoffoni, 2013).
California will likely have more frequent droughts and continued species invasions that may lead to trade-offs that balance the selective pressures of opposing environmental filters (Ishida et al., 2008;Pierce et al., 2017;Seebens et al., 2015).
Strategies such as drought escape, avoidance, and tolerance are coordinated by physiological and morphological traits, and can be used to further understand plant responses to global change (Kooyers, 2015;Levitt, 1980). Drought tolerance and escape are more consistent with the classic leaf economic spectrum theory, while drought avoidance coordinates characteristics not typical of the leaf economic spectrum (Kooyers, 2015;Sandel et al., 2021;Volaire, 2018;Wright et al., 2004). Drought tolerance is more common for woody species with conservative traits (Ingram & Bartels, 1996;Volaire, 2018). Drought escape and avoidance are more common for herbaceous species with acquisitive traits that have active growth during periods of high soil water availability, distinct from drought-tolerant species that can maintain growth during periods with low soil water (Huang et al., 2018;Kooyers, 2015;Welles & Funk, 2021). Drought escape is common for annuals and is typified by quick growth and high fecundity (Huang et al., 2018). Drought avoidance is prevalent for both annuals and perennials, and these species rely on high WUE, limited vegetative growth, and high root:shoot ratio (Kooyers, 2015;Levitt, 1980). Competitive release results in increased fitness or productivity for a species when its competitor is removed or negatively affected by environmental conditions (Menge, 1976;Segre et al., 2016). California plants may experience competitive release during drought because their invasive counterparts respond more negatively to drought compared to native annuals in greenhouses and perennials in situ (Luong et al., 2021;Valliere et al., 2019). Certain native perennial bunchgrasses are able to withstand competition from invasive species (Corbin & D'Antonio, 2004), but less is known about other life-forms. California species that are affected by invasion have lower aboveground productivity and some species adjust leaf traits associated with competitive ability to maximize fitness (Drenovsky et al., 2012;Seabloom et al., 2003). Yet, how invasive competition and drought interact to drive plant growth, morphology, and competitive release is less understood (Poorter et al., 2012;Segre et al., 2016).
We tested how drought and invasive competition shape functional traits and biomass allocation for five California grassland species commonly used for restoration in central California. In a controlled glasshouse environment in Santa Cruz, CA, USA, we measured physical traits (biomass, growth rates, specific leaf area, leaf area, major vein length per unit area, leaf lobedness, leaf C:N, and δ 13 C) and photosynthetic gas exchange rates (A net , g s ) of native species experiencing episodic drought and invasive competition.
Environmental filter theory predicts that plants will grow slower under drought, so we hypothesized droughted plants would have reduced instantaneous leaf-level gas exchange, and also greater root allocation due to optimal partitioning. We predicted that competition would lead to changes in leaf traits to acquire space and light resources. We also hypothesized native species would exhibit tradeoffs that fall on a spectrum related to resource conservatism (high VLA, lobedness, iWUE, and C:N; see methods) versus acquisition (high SLA, ARGR, A net , and leaf N) observed via functional traits in response to factorial drought and competition, as predicted by the leaf economic spectrum and environmental filter theory.

| MATERIAL S AND ME THODS
The five native species in this study were chosen because they are commonly used for grassland restoration in California (Table 1; Jepson eFlora, 2020). We selected the five invasive species (Table 1) based on their high cover from previous vegetation surveys (Luong et al., 2021). The invasive species are regionally ubiquitous and monitored by the California Invasive Plant Council (www.cal-ipc.

| Experimental design
We set up a two-way factorial study manipulating drought and competition from invasive species in a rooftop glasshouse at the University of California, Santa Cruz, between October 2019 and April 2020. In October 2019, we sowed seeds of native species (Table 1) on PRO-MIX high porosity soil (6:1:1 of sphagnum peat moss, perlite, and limestone) in seedling flats partitioned by species.
Seedlings were kept well watered and then healthy seedlings similar in size from each species were individually transplanted into 32 4.5-L growing containers (17 cm tall × 16 cm diameter). Transplanting occurred at least 2 weeks after germination and after plants developed two sets of true leaves. Once transplanted, the native plants were well watered and unfertilized for 6 weeks. Because most fertilizers are water based, droughted plants could not be fertilized, so all plants were kept unfertilized. We randomized pot locations on the glasshouse tables weekly to limit microclimate effects. Average daytime temperatures and relative humidity (RH) were 16.5°C and 68.1% while nocturnal conditions were an average of 10.7°C and 78.4% RH. Proportions of light-to-dark hours started at 11 h light to 13 h dark in October 2019, slowly decreased to its minimum in December, with 9.5 h light to 14.5 h dark, and increased to reach 13 h light to 11 h dark at the end of the study in April 2020. We did not augment the light intensity or cycle.
Eight replicates of each species were assigned to treatments within a 2 × 2 factorial design: (1) well watered (no manipulation); (2) episodic drought; (3) invasive competition; and (4) invasive competition and episodic drought simultaneously. We harvested three replicates from each native species in each treatment group to determine baseline aboveground and belowground biomass during week 6, leaving five replicates per species in each treatment.
On week 6 we sowed five common invasive species (Table 1) in half of all pots to establish the competition treatment. We sowed invasives at densities based on historic field surveys (Heady, 1977; 185 mg per pot C. pycnocephalus, 100 mg F. bromoides, 103 mg G. dissectum, 85 mg M. polymorpha, and 69 mg for R. sativus) corrected for the surface area of a 4.5-L pot (201 cm 2 ). On week 8, we applied an episodic drought (Duan et al., 2014) where water was withheld until a minimum stomatal conductance (g s ; see list of abbreviations in Table 2) occurred for native species in an initial and secondary drought period (g s <0.05 mol m −2 s −1 H 2 O). Rehydration occurred concurrently for all individuals of the same species after half of the individuals droughted from that species reached the minimum g s threshold. The g s was measured for all native individuals using an open-mode portable photosynthesis system (Model LI-6400; Li-Cor, Inc.). Droughted plants were then rehydrated to pot capacity for 10 days, then exposed to a second drought. This episodic drought protocol with two drought periods has been shown to result in plant glasshouse drought responses that best mimic in situ plants (Duan et al., 2014). Due to interspecific variation in stomatal conductance to episodic drought (Table S1), the duration of drought varied for each native species. No native species had premature mortality.
Non-natives used for the competition treatment persisted through the drought to the end of the experimental period (Table S1)

| Functional traits
Traits were only sampled from native species. We collected three replicates of biomass from each species and treatment group prior to any treatments (week 6) and for all remaining individuals after the second episodic drought. We cut each plant at the base of the soil where the shoots and roots were differentiated. We washed soil out of the belowground biomass samples by gently dunking them in a series of four buckets with gentle agitation by hand. After the final bucket, we ran water over the roots to remove any remaining silt or perlite while over a 500 µm sieve to prevent root loss. We saved roots that broke off while washing to be included in dry biomass weights and estimated a loss of approximately 5% of total root biomass. Samples were dried at 60°C for at least 72 h before quantifying aboveground (AGB) and belowground biomass (BGB). We calculated aboveground relative growth rates (ARGR) and belowground relative growth rates (BRGR) by subtracting the final biomass of an individual by the baseline average taken in pretreatment (week 6), divided by the total growing days ( Table 2).
We sampled leaves from native plants prior to any treatments and at the end of the second drought to quantify effects on specific leaf area (SLA), major vein length per unit area (VLA), leaf lobedness, leaf C:N, and δ 13 C (see list of abbreviations in Table 2). Pretreatment leaf characteristics and biomass were used to confirm there was no grouping effect prior to experimental treatments (p all > .05). SLA is related to photosynthetic ability, palatability, leaf life span, and growth rates (Sandel et al., 2021;Wright et al., 2004). SLA often decreases in response to drought but increases due to competition (Wright et al., 2004). Total leaf area is associated with competitive ability because it is related to light capture, shading, water loss, and energy budgets (Liu & Stützel, 2004;Pérez-Harguindeguy et al., 2016). Increased VLA can improve drought resistance by increasing vein reticulation and redundancy for water and sugar transport (Sack & Scoffoni, 2013). Leaf lobedness affects the leaf energy balance and is calculated as the ratio of leaf perimeter squared to the product of leaf area and π (Cadotte et al., 2015;Luong et al., 2021). Grass leaves may not be dissected but operationally, can have high leaf lobedness because of their high leaf perimeter:area ratios. Increased leaf lobedness decreases the effective length that wind travels at the leaf surface and reduces the boundary layer, resulting in increased cooling via conduction and convection, potentially decreasing leaf-level transpiration (Lambers et al., 2008). Leaf C is related to palatability and leaf N to photosynthesis (Pérez-Harguindeguy et al., 2016). Plants with high C:N values are often more resistant to drought but may be less competitive than plants with low leaf C:N (Drenovsky et al., 2012;Pérez-Harguindeguy et al., 2016). δ 13 C is often used as a proxy for WUE (Table 2) because they are correlated for most species (Lajtha & Marshall, 1994

| Analyses
All analyses were completed with R statistical software (Version 4.0.4; R Development Core Team, 2007). We ensured data had a Gaussian distribution and equal variances before using parametric tests. We used different statistical tests depending on the hypothesis to be tested. Data were processed and visualized with plyr, cowplot, and gg-plot2 (Wickham, 2020;Wickham et al., 2018;Wilke, 2020).
Because PLA, PRA, and ARR were only measured for individuals that experienced drought, the differences between droughted individuals with or without invasive competition were analyzed using t-tests. Traits (SLA, VLA, lobedness, C:N, δ 13 C, and root:shoot biomass) collected at the end of the second drought period were compared using two-way analysis of variance (ANOVA) to test for interactive effects of drought and invasive competition. Competitive release was defined on a physiological basis where there was greater iWUE, ARR, PRA, or lower PLA during combined drought and competition, compared to when plants were exposed to drought with no competition (Segre et al., 2016). For data collected weekly (A net , g s , and iWUE), we used mixed linear models with time as a fixed variable to test for the effects of drought and competition over time. We used a regression to test for a correlation between δ 13 C and iWUE.
We used a principal component analysis (PCA) to detect tradeoffs between measured traits along a spectrum of two principal components (PC) using the vegan package (Ishida et al., 2008;Oksanen et al., 2018;Pierce et al., 2017). PCA can be used to decrease dimensionality in multivariate trait space by compressing multiple variables into fewer selected intercorrelated axes (principal components). Trait values were then tested for correlations against main PCs to determine intertrait relationships (Pierce et al., 2017; Table   S2)

| Photosynthetic gas exchange
Midday A net and g s of B. carinatus, D. aurantiacus, and L. nanus were negatively affected by drought, and further reduced for L. nanus through an interaction with competition (Table 3, Figure S2F-J).
Drought decreased iWUE for D. aurantiacus and L. nanus, and was further limited by an interaction with competition for L. nanus.
Diplacus aurantiacus had an interactive effect, resulting in higher iWUE for droughted plants only when experiencing competition (Table 3). Aside from interactions with drought, invasive competition did not affect leaf gas exchange. Midday A net ( Figure S2A-E) had a significant and negative reduction over time for all species except B. carinatus, whereas g s decreased over time for all species but B. carinatus and S. malviflora (Table 3). iWUE had an inverse relationship with time for all species, except for L. nanus, which had greater iWUE over time, and S. malviflora which had no relationship with time ( Figure S2K-O). Midday iWUE was positively correlated with leaf δ 13 C of native species (p = .016; R 2 = .51; Figure S3).
Invasive competition increased nocturnal respiration for D. aurantiacus (p = .008) and for S. pulchra facing drought and competition simultaneously (p = .010), but no other species (Table S1; Figure   S4). Nocturnal respiration was not affected for study species when only facing drought (p all > .05). Nocturnal stomatal conductance was negatively affected by drought for D. aurantiacus (p = .040), L. nanus (p < .001), and S. pulchra (p = .004). Nocturnal stomatal conductance of L. nanus was further reduced by invasive competition in drought conditions (p = .012).

| Trade-offs in growth responses
We found that most traits grouped along two principal components (PC) that explained 40.3% and 22.4% of trait variance (Figure 4).
Variances were not partitioned by treatments, but instead by species identity. PC1 was related to resource acquisition versus conservatism, which Kooyers (2015) related to strategies for drought escape versus tolerance (Kooyers, 2015). The acquisition end of the axis was correlated with high SLA, growth rates (ARGR and BRGR), midday A net , and leaf %N. The resource conservative end of PC1 was related to high leaf C:N, VLA, and leaf lobedness (Table S2). PC2 was driven by trade-offs related to above-versus belowground growth allocation. Allocation of resources belowground was associated with high root:shoot, iWUE, and δ 13 C, which contrasted with aboveground growth strategies that were correlated with high ARGR and leaf %C (Table S2). Nocturnal leaf respiration, nocturnal g s , and midday g s were not strongly related to either axis.

| DISCUSS ION
Most greenhouse-grown native coastal grassland C 3 species that we studied exhibited drought-adapted trait adjustments and a limited amount of adjustments for competition. Our hypothesis that leaf gas exchange would be more affected by drought and less so by competition, and morphological leaf traits more to competition than drought was supported. Moreover, we found evidence (described below) that D. aurantiacus may experience competitive release during drought. Although it has been shown that drought in California can more negatively affect invasive species than natives, this may be the first evidence to show California species experiencing competitive release in a controlled environment. In support of our predictions and consistent with environmental filter theory, we found trade-offs between leaf trait conservatism versus acquisition.
However, we also found trade-offs related to belowground versus aboveground allocation within the multivariate trait space, consistent with optimal partitioning theory.

| Invasive competition
According to optimal partitioning theory, increased allocation to roots in response to competition for L. nanus, S. malviflora, and S. pulchra suggests that belowground resources may be more limiting than light or aboveground space for these California coastal grassland species (Bloom et al., 1985;Poorter et al., 2012;Rehling et al., 2021). Aside from biomass allocation, we found certain species adjusted functional traits in response to competition. Bromus carinatus exhibited more acquisitive leaf traits (e.g., higher SLA), had more developed root systems to support higher resource needs, and recovered photosynthesis more quickly after drought when undergoing competition from invasives, indicating that this species may be useful for ecological restoration of heavily invaded areas. Lupinus nanus had lower leaf area and SLA, but higher VLA and lobedness in competition, which could indicate its sensitivity to competition.

| Invasion during drought
Although S. pulchra increased root:shoot allocation in response to drought as predicted by optimal partitioning theory, D. aurantiacus showed an opposite response (Poorter et al., 2012). But D. aurantiacus can become woody over time, so investing resources aboveground could provide some degree of drought tolerance (Domec et al., 2017) and enhanced support to compete for light (Sun et al., 2003), and in this regard, responses are consistent with optimal partitioning. Increased δ 13 C and iWUE during drought are consistent with upregulated drought tolerance (Lajtha & Marshall, 1994), Note: Treatment effects were compared using generalized linear models with a fixed time effect (based on weekly measurements). A net = net CO 2 assimilation; g s = stomatal conductance; iWUE = intrinsic water-use efficiency; N = 5 for all groups. All treatments were pooled to test for time effects, significance indicates change over time. Graphical representation (and direction of change) of these findings can be seen in Figure S2.
F I G U R E 3 (a) PLA (the percent loss of assimilation) and (b) ARR (the assimilation recovery rates) of native species with competition from invasive species (yellow) or not (blue). *Denotes significant pairwise differences due to competition based on t-tests. The colored bar = interquartile range; the solid line in the bar = median; lines extending out of bar = upper and lower quartile range; and points = outliers related to resource acquisitive strategies (Funk et al., 2008;Wright et al., 2004) and possibly underlies drought escape (Kooyers, 2015), especially for plants in semi-arid environments. Indeed, other acquisitive traits (A net , ARGR, BRGR, and %N) responded similarly to SLA in response to factorial drought and competition. Drought tolerance appears to be the strategy used by D. aurantiacus, as it often actively grows through the summer months and had more resource conservative traits (higher C:N and δ 13 C). The pattern of trait relationships within the resource acquisitive versus conservative spectrum is consistent with environmental filter theory, whereas the trade-offs in above-and belowground allocation support optimal partitioning theory (Bloom et al., 1985;Funk et al., 2008).
In general, leaf gas exchange was negatively affected by drought and time, but not competition which supports environmental filter theory's prediction that growth will be more conservative during harsh conditions (Funk et al., 2008). Typically, physiological processes respond in shorter time scales compared to leaf morphology because physiological mechanisms are often molecular (Lambers et al., 2008), which may explain why gas exchange responded to drought.
Physiological leaf traits (leaf C:N and δ 13 C) were also primarily affected by drought and not as much by competition. Competition can have mixed effects depending on whether the invader is a stronger above-or belowground competitor (Poorter et al., 2012). Similarly, we found that native species exhibited morphological leaf trait (SLA, VLA, and lobedness) adjustments more often to competition, but in certain cases to drought. This response is consistent with optimal partitioning whereby individuals obtain limited aboveground light and space resources (Bloom et al., 1985;Drenovsky et al., 2012).
In other instances, morphological traits were responsive to competition, and in a few cases to drought (Poorter et al., 2012). We also note that photosynthesis can decrease as plants age and do not need to compete for space as much as when they are younger (Stromberg et al., 2007).
Diplacus aurantiacus showed evidence of competitive release.
Because certain invasive species respond more negatively to resource limitation compared to some California natives ( These relationships are consistent with optimal partitioning and environmental filter theories (Bloom et al., 1985;Funk et al., 2008;Poorter et al., 2012).
Our results have management implications for California grassland restoration and native habitat management. Because certain native species were more resilient or resistant to drought (B. carinatus, S. malviflora, and S. pulchra) and others were more sensitive (L. nanus), it may be resource effective for restorationists to use drought-adapted species if planting during extended drought periods, and limit introducing greater species richness to wetter years. Some may also consider using supplemental irrigation if sensitive species must be planted (Stromberg et al., 2007). Bromus carinatus exhibited beneficial trait adjustments for higher competitive ability, indicating it may be ideal to use in invaded areas. Diplacus aurantiacus showed evidence of competitive release, suggesting that these species will require less invasive species control during drought periods.

CO N FLI C T O F I NTE R E S T
Authors declare no conflict of interests. Writing -original draft (supporting).

DATA AVA I L A B I L I T Y S TAT E M E N T
Plant trait data were deposited in the TRY-TRAIT database. Data presented are available (including trait data on TRY-TRAIT) on PANGAEA Data Publisher for Earth and Environmental Sciences (Luong & Loik, 2022).

S U PP O RTI N G I N FO R M ATI O N
Additional supporting information may be found in the online version of the article at the publisher's website.